APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1994, p. 357-3620099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Pseudomonas putida Strains Which ConstitutivelyOverexpress Mercury Resistance for Biodetoxification of

Organomercurial PollutantsJOANNE M. HORN,* MAREN BRUNKE, W.-D. DECKWER, AND KENNETH N. TIMMIS

National Research Center for Biotechnology (GBF), 3300-D Braunschweig, Gennany

Received 6 July 1993/Accepted 31 October 1993

Improved biocatalysts for mercury (Hg) remediation were generated by random mutagenesis of Pseudo-monas putida with a minitransposon containing merTPAB, the structural genes specifying organomercuryresistance. Subsequent selection for derivatives exhibiting elevated resistance levels to phenylmercury allowedthe isolation of strains that constitutively express merTPAB at high levels, conferring the ability to cleave Hgfrom an organic moiety and reduce the freed Hg(II) to the less toxic elemental form, Hg°, at greater rates.Constitutive overexpression ofmerTPAB had no apparent effect on culture growth rates, even when Hg(II) wasinitially present at otherwise toxic concentrations. These properties were also combined with benzene andtoluene catabolism, allowing detoxification of the metal component of phenyl mercuric acetate, as well as

degradation of its aromatic moiety.

Mercury (Hg) is a toxic metal that has been released intothe environment in substantial quantities (27). Mercurytoxicity results from the capacity of Hg in its bivalentcationic form [Hg(II)] to bind sulfhydryl, thioether, andimidazole groups and thereby inactivate enzymes (26). Or-ganic species of mercury, both alkyl and aromatic deriva-tives, are capable of accumulating in the tissues of higherorganisms, where they cause systemic disease (17).

Since the total amount of extant Hg is finite and unchange-able, remediation of Hg pollution can be aimed only ataltering its ionic form to a less toxic species and/or seques-tering it, ideally in forms which can be recycled for furtheruse. Recently developed remediation strategies are aimed atreducing Hg(II) to the more inert, volatile elemental form(Hgo) by employing the mechanism of bacterial Hg resis-tance (Hgr), which relies on the activity of a cytosolicmercuric reductase (6, 9, 34).Broad-spectrum bacterial Hgr involves the mercuric re-

ductase, an Hg-specific transport system, and an organomer-curial lyase that protonolytically cleaves carbon-Hg bonds(2, 24, 34, 38). The broad-spectrum Hgr systems, through thesuccessive activities of the lyase and the reductase, thusprovide resistance to both organomercurial compounds andmercurial ions.

Hgr structural genes are encoded in a single operon that isregulated primarily by the merR gene product, which is itselftranscribed contiguously to, but in the opposite directionfrom, the merTPABD operon (16, 25, 30). The merT andmerP gene products are involved in Hg(II) uptake andtransport (15), merA specifies the mercuric reductase, andmerB encodes the organomercurial lyase (see reference 37for a review). merD, the most promoter-distal gene identi-fied, has been associated with a transcriptional coregulatoryfunction (22, 29).Here, we report the generation and selection of Pseudo-

monas putida strains with heightened mercurial detoxifica-tion properties as a result of their constitutive overexpres-

* Corresponding author. Present address: Center for Environ-mental Diagnostics and Bioremediation, University of West Florida,Pensacola, Fla. 32514. Phone: (904) 474-2648. Fax: (904) 474-3130.

sion of the merTPAB genes. The ability to reduce Hg(II) atincreased rates in higher concentrations of mercurials was

genetically combined with a benzene degradative pathway.Some derivative strains were thus able to dissociate thechemically dissimilar components of an organomercurialcompound, phenylmercuric acetate (PMA), into its metaland aromatic elements and separately detoxify each.

Bacterial strains and growth conditions. The bacterialstrains and plasmids used in this study are listed in Table 1.L medium and low-phosphate (100 ,uM) 121-salts minimalmedium (LP121) were prepared as previously described (14,19). M9 and M63 minimal media (19) were both supple-mented with 0.2% citrate as a carbon source. 0.001%Pseudomonas stock salts (1) additionally supplemented min-imal M9 media; 0.2% Casamino Acids (Difco) were added toM63 minimal medium.

Construction, selection, and screening of highly PMA-resistant (PMAr) P. putida derivatives. Overexpression ofbroad-spectrum mer determinants could theoretically resultin heightened levels of resistance to organomercurials,thereby providing a basis for the direct selection of overex-

pressing derivatives. The utility of this approach was testedwith P. putida by using plasmid pUTHg, a narrow-host-range replicon that encodes the merTPAB broad-spectrummercury resistance derived from pDU1358 (12). The pUTHgmerTPAB genes are flanked by the inverted repeats of TnS,which have been abbreviated to 19 bp, and merR and merDare absent from the construct (12, 13).Two strains of P. putida were chosen as recipients of

pUTHg. P. putida Fl is a naturally occurring soil isolate thatharbors a chromosomally encoded pathway for the catabo-lism of benzene (11). P. putida KT2442 is a rifampin-resistant derivative of an isolated soil organism that isincapable of catabolizing benzene (18).Transposon-containing plasmids were transferred from

Escherichia coli donor C600(pFRC37p) or SMlOApir(pUTHg) into either P. putida KT2442 or P. putida Flrecipients by filter mating (7). Selection for plasmid mobili-zation and transposition were performed on M9 or M63media containing 1.5 or 6 ,ug of HgCl2 per ml (and 50 ,ug ofrifampin per ml for P. putida KT2442 recipients). Excon-

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TABLE 1. Bacteria and plasmids

Strain or plasmid Description Reference

BacteriaE. coliHB101 proA2 leuB6 thi-1 lacYl hsdR hsdM recA13 supE44 rpsL20 3C600 supE44 hsdR thi-1 thr-I leuB6 lacYl tonA21 39SM1O xpir thi-1 thr leu tonA lacYsupE recA::RP4-2-Tc::Mu Kmr Xpir 20

P. putidaKT2442 hsdR Rift Tol- Ben- 18Fl todFCIC2BADEJ+ Tol+ Ben' 11KT2442::Tn501-132 hsdR merTPAD+ Hgr Rif' Tol- Ben- This studyKT2442::mer hsdR merTPAB+ PMAr Hgr Rift Tol- Ben This studyFl::mer todFCIC2BADEJ+ merTPAB+ PMA' Hgr Tol+ Ben' This study

PlasmidspRK2013 on ColE1+ tra RK2+ Kmr 7pFRC37p on ColE1+ mob RP4+ TnS01 merR+ merTPAD+ Apr Ben- F. RojopUTHg on R6K+ mob RP4+ merTPAB+ AmerDR Apr Hgr 13pGP704 on R6K mob RP4, multiple cloning site of M13tgl31, Apr 20

jugants were screened on either M9 media with rifampin and6 ,ug of HgCl2 per ml for mobilization and transposition ofTn5OI or M63 media supplemented with 10 ,ug of PMA perml for recipients of pUTHg matings.

Transposition frequencies of the merTPAB genes in theP. putida recipients were found to be in agreement withthose observed previously (13). We reasoned that mini-Tn5merTPAB insertions downstream of proximal host promot-ers could result in increased mer expression, with a coinci-dent rise in Hg and PMA resistance (Hgr PMAr), eventhough increased mer expression has not been correlatedwith increased resistance in E. coli (23, 25, 31). One thou-sand twenty-five P. putida KT2442 and 800 P. putida Fltransconjugant isolates were patched onto L medium con-taining 10 ,ug of PMA per ml, and subsequent selection ofrecipient isolates with enhanced Hgr PMA' was accom-plished by stepwise screening of these P. putida KT2442 andFl transconjugants on L media containing from 50 to 250 jigof PMA per ml. Screening on PMA avoided the false-positive background resulting from screening on HgCl2(unpublished observation).

All examined P. putida KT2442 and Fl transconjugantsgrew on media containing 50 jig of PMA per ml. Some P.putida KT2442 transconjugants also showed growth onmedia containing up to 80 ,ug of PMA per ml, while a subsetof P. putida Fl isolates showed growth on media containingup to 250 ,ug of PMA per ml. Comparatively, 10 ,ug/ml haspreviously been used as a MIC of PMA (13).

Representative individual transconjugant strains (20 P.putida KT2442 and 20 P. putida Fl strains) were found to besensitive to carbenicillin (500 ,ug/ml), piperacillin (30 ,ug/ml),and kanamycin (30 ,ug/ml), demonstrating that there hadbeen no replication or integration of either donor plasmid inthe examined recipients and providing preliminary evidenceof mini-TnS transposition into the host chromosomes(::mer).

Verification of stable merTPAB transposition. P. putidaKT2442::mer (isolates 67, 73, 2-4, 2-49, and 2-121) andFl::mer (isolates 1-1, 1-7, 1-22, 10, and 13) isolates wereanalyzed by Southern hybridization (35) to verify the trans-position of the mer operon and assess the uniqueness oftransposition events. Genomic and plasmid DNAs were

purified (32, 33), restriction enzyme digested, and blotted toBiodyne B membranes, which were then hybridized accord-ing to the recommendations of the manufacturer (Pall Bio-support). DNA fragments used as probes were isolated byusing GeneClean (Bio 101, Inc.), and all probes were labeledin vitro with [t_-32P]dCTP by random primer extension(Amersham).The results showed that when probed with an SfiI-gener-

ated 3.2-kb pUTHg-derived merTPAB sequence, a corre-sponding fragment hybridized in all like-digested P.putida::mer genomic DNA samples and KT2442 TnSOI-containing DNA (4, 5, 13, 21), demonstrating that themerTPAB operon-containing fragment is indeed present inthese isolates. Parental KT2442 and Fl strains showed nohybridization with the mer probe (Fig. 1).When the same set of DNAs were subjected to hybridiza-

tion with the mer cloning vector pGP704 (13, 20), only thepUTHg control demonstrated positive hybridization (datanot shown). Thus, the presence of merTPAB in the P.

A.

Kb

23.1-

94-6.6-44-

2.3-2.0-

B.

Kb 1 2345678 9

233-1'.-~6.8

4.4-A

J *,,I

2.3-

FIG. 1. Southern hybridization analysis of P. putida::merstrains. Blots were probed with a labeled pUTHg-derived merT-PAB-containing fragment (3.2 kb). (A) P. putida::mer KT2442strains. Lanes: 1, P. putida KT2442 (SfiI); 2, isolate 67 (Sfi1); 3,isolate 73 (SfiI); 4, isolate 2-4 (Sfi1); 5, isolate 2-49 (SfiI); 6, isolate2-121 (SfiI); 7, pUTHg (SfiI); 8, isolate 67 (EcoRI); 9, isolate 73(EcoRI); 10, isolate 2-4 (EcoRI). (B) P. putida::mer Fl strains.Lanes: 1, P. putida Fl (SfiI); 2, isolate 1-1 (SfiI); 3, isolate 1-7 (Sfi1);4, pUTHg (Sfi1); 5, isolate 1-22 (SfiI); 6, isolate 10 (SfiI); 7, isolate 13(Sfi1); 8, P. putida::mer KT2442::Tn501-132 (AvaI); 9, pUTHg(EcoRI).

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KT2442::Tn5Ol -132

KT2442::mer -73

KT2442::mer -2-4

KT2442::mer -2-4

Q.

Q:

KT2442::mer -2-116

KT2442::mer -2-121

Fl ::mer -10

F1::mer- 13

Fl ::mer-1-1

Fl ::mer -1-7

Fl ::mer-1-22

6 l0 20 30 40 50 60 80Maximal HgCI 2or PMA resistance (jg/ ml)

FIG. 2. Maximal HgCl2 and PMA resistance of P. putida::merisolates. The greatest concentrations of PMA and HgCl2 in solid (-and M, respectively) or liquid (U and 1, respectively) LP121 mediathat still permitted growth of P. putida::mer strains are shown. Tenand 15 ,ug of PMA per ml in LP121 liquid and solid media,respectively, prevented the growth of P. putida KT2442::Tn501-132and parental strains P. putida KT2442 and Fl. Parental-straingrowth was also inhibited by HgCl2 at concentrations of 6 and 35p.g/ml in liquid and solid LP121 media, respectively.

putida::mer derivatives was not due to cointegration butdemonstrated true transposition events.

Hybridization of the mer probe to EcoRI-digested P.putida::mer DNA, for which there is a single site in themini-TnS merTPAB construct (13), resulted in two hybridiz-ing fragments per isolate; all fragments were of dissimilarmobilities (KT2442 derivatives are shown in Fig. 1). Thus,mer transposition in these strains occurred once and resultedin integration in a single, unique host sequence.

Determination of upper levels of resistance to HgCl2 andPMA in minimal media. The highest concentrations of HgCl2and PMA which still allowed growth in both solid and liquidLP121 were determined for the P. putida::mer strains (rep-resentative results are shown in Fig. 2). P. putidaKT2442::TnS01-132, which contains a stable insertion ofTnSOJ (encoding a narrow-spectrum mer operon), provided acomparative measure of prototypical Hgr and expressionfrom a native mer promoter (4, 16, 21, 28, 36).

Resistance levels on solid media were indicated by thegrowth of isolated colonies on LP121 plates containing eitherHgCl2 or PMA (Fig. 2). Upper levels of resistance in liquidmedia were determined by growing strains at 30'C to mid-logphase (A60 = 0.5) in liquid LP121; then, HgCl2 or PMA wasadded to the final concentration (except for KT2442::TnSOl-132, which was induced with 0.8 ,ug of HgCl2 per ml at an

A600 of 0.5 and then incubated for 1 h, at which time HgCl2was added to the final concentration). The A6. was deter-mined after approximately 4 h of growth in HgCl2 or PMAand compared with the A600 of parallel cultures grown underhe same conditions without the addition of either HgCl2 or

TABT E 2. Mercuric reductase activities of HgCl2-induced oruninduced P. putida::mer cultures

Mercuric reductase activityaP. putida strain or

isolate Without WithHgCl2 HgCI2

KT2442 <0.2 NDb

Fl 0.2 ND

KT2442::TnS01-132 <0.2 12.1

KT24j2::mer37 7.0 8.145 14.5 12.767 26.0 29.073 48.3 44.82-1 9.7 8.22-3 14.3 18.72-4 29.0 32.42-49 27.3 23.42-65 12.7 11.62-84 12.7 15.12-116 15.9 17.32-121 28.7 31.0

Fl: :mer10 34.2 35.412 7.7 7.913 33.3 36.318 10.1 9.831 29.4 31.138 12.8 11.51-1 35.8 39.61-6 13.3 14.21-7 35.0 35.91-14 27.1 28.21-22 34.0 34.6a HgCl2-dependent NADPH oxidation (micromolar NADPH oxidized per

min per mg of protein) (see the text). The data are averages for three separatetrials.bND, not determined.

PMA. Resistance was scored as approximating the turbidityof the untreated control.

Eighty-five percent of the 20 P. putida KT2442::merisolates tested demonstrated a 12.5 to 37.5% increase in thelevel of Hgr compared with that for P. putida KT2442::TnS01-132 when grown on solid LP121 media. However,only 30% of these strains demonstrated any increase in Hgrwhen grown in liquid LP121; the levels were all 28.5%greater than those for P. putida KT2442::Tn501-132 in thesecases. When the 20 P. putida KT2442::mer isolates weretested in PMA-containing LP121, they showed higher resis-tance over a shorter range of PMA concentrations on solidmedia than in liquid media.

Generally, the 20 P. putida Fl::mer strains demonstratedgreater Hgr than the P. putida KT2442::mer strains that weretested. Fifty percent showed a 14.3 to 85.7% increase in Hgrcompared with the levels for P. putida KT2442::TnS01-132on solid and liquid LP121 media. However, upper levels ofPMAr for mer derivatives of P. putida KT2442 and Fl werecomparable (Fig. 2).mer gene expression in P. putida::mer isolates. The enzy-

matic activity of the merA gene product, mercuric reductase,was used to quantify mer gene expression. Mercuric reduc-tase assays were conducted on extracts of 12 representative

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2.0 P. putida F1::mer-1-1

1.6

E1.2F

0.8

0.4

0.0

0 2 4 6 8TIME (hr)

10 12

6 8 10 12 0 2 4 6 8 10TIME (hr) TIME (hr)

4 6 8 10 12TIME (hr)

FIG. 3. Growth of P. putida strains in the presence of HgC12. LP121 medium containing 6 ,ug of HgC12 per ml (-), 6 ,ug of HgCl2 per mladded at anA600 of 0.5 (A), or no added HgCl2 (0) was inoculated with a stationary-phase culture and incubated at 30°C. P. putida::Tn501-132was preinduced with 10 ,uM HgCl2 prior to addition of HgC12 at mid-log phase. Enumeration of viable cells was used to confirm the results.

P. putida KT2442::mer and 11 P. putida Fl::mer isolatesgrown at 30°C in LP121 to an A600 of 0.5, at which pointeither they were allowed to continue growing without theaddition of HgCl2 or HgCl2 was added at the highest con-centration still allowing growth. The TnS01-containing con-trol strain was induced with 0.8 ,ug of HgCl2 per ml for 2 h,after which HgCl2 was added to 6 ug/nml. Cultures weregrown to a final A600 of 1.5 and then harvested. Crudeextracts were assayed for mercuric reductase activity by themethods of Fox and Walsh (9) and Schottel (34) by monitor-ing the rate of HgCl2-dependent oxidation of NADPH.

P. putida KT2442::TnS01-132 merA expression in theabsence of HgCl2 was undetectable (Table 2). As had beenfound previously in studies using E. coli hosts, TnSOJ-encoded merA expression is Hg(II) inducible, because of theactivity of the MerR protein (16, 25, 30). In contrast, all 23 P.putida::mer isolates tested demonstrated approximately thesame mercuric reductase activities with or without theaddition of HgCl2 (Table 2). merTPAB expression in thesestrains is, therefore, constitutive; Hg(II) is not required fortheir expression, presumably because of the deletion ofmerR (8, 12, 16, 25).The level of constitutive mercuric reductase activity dis-

played by the P. putida::mer isolates varied significantlyfrom the induced MerA activity shown by P. putidaKT2442::TnS01-132. P. putida KT2442::mer isolates dis-played up to a 3.7-fold increase (isolate 73) and P. putidaF1::mer strains showed a maximal 3.3-fold greater reductaseactivity (isolate 1-1) than the Tn501-containing strain (Table2).Growth rate determination in the presence of mercury.

Growth curves for the parental Hg-sensitive (Hgs) P. putidaKT2442 and Fl strains and their KT2442::mer-73,Fl::mer-1-1, and KT2442-TnS01-132 Hgr derivatives weregenerated in LP121 media to assess the effect of merTPABoverexpression on culture growth rate.

Overexpressing merTPAB strains showed no significanteffect of Hg exposure on growth rate when inoculated inLP121 media (1:100 with a stationary-phase culture) contain-ing 6 p,g of HgCl2 per ml, while both parental P. putida andthe TnSOJ derivative were unable to grow under identicalconditions (Fig. 3). P. putida::TnS01-132 Hgr is Hg(II)inducible (37); therefore, exposure to toxic concentrations ofHg(II) presumably prevents cell growth before mer induc-tion can occur, demonstrating an Hgs phenotype. However,constitutive overexpression ofmerTPAB conferred Hgr evenat initial concentrations of Hg(II) that were otherwise toxic(Fig. 3).When 6 ,g of HgCl2 per ml was added at mid-log phase,

there was again no appreciable effect on the growth rate ofKT2442::mer-73 or Fl::mer-1-1, while the Hgs parentalcontrol cultures showed an immediate cessation of cellgrowth and an induced P. putida KT2442::TnS01-132 culture(10 ,uM HgCl2 added 2 h prior to the addition of 6 ,ug/ml atmid-log phase) showed a slightly enhanced growth rate (Fig.3).Growth and dioxygenase activity of P. putida Fl::mer iso-

lates on PMA. Since P. putida Fl::mer isolates harbor boththe transposon-encoded organomercurial lyase and a chro-mosomally specified pathway for benzene degradation (11),they are theoretically capable of both cleaving Hg(II) fromPMA and utilizing the generated benzene moiety as a solesource of carbon and energy. The ability of 21 P. putidaFl::mer isolates to grow on solidified M63 minimal mediawith 160 ,ug of PMA per ml provided as the sole source ofcarbon was assessed. Isolated colonies which arose weretested for activity of one of the enzymes involved in benzenedegradation, catechol 2,3-dioxygenase, by colorimetric as-say (10).Both the P. putida Fl and the P. putida KT2442::mer-73

controls were unable to grow on PMA and showed nodioxygenase activity. P. putida Fl lacks broad-spectrum Hgr

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and, so, is sensitive to PMA (PMAS) (Fig. 1). Furthermore,since P. putida Fl encodes no organomercurial lyase and itsaromatic degradative pathway might not accept PMA as asubstrate, it may be unavailable as a carbon source to thisorganism. P. putida KT2442::mer-73, on the other hand,does encode PMA' (Fig. 1) and, thus, can generate benzenebut could not grow on PMA because of its lack of a benzenedegradative pathway.Nine of the 21 P. putida Fl::mer strains tested were,

however, able to grow and showed dioxygenase activity onM63 PMA media. Lack of growth on PMA by the remainingFl::mer isolates might be explained by comparatively lowlevels of mer expression in these strains, but no suchcorrelation was apparent. Alternatively, the induction ofbenzene catabolism or levels of expression of these deter-minants may be altered in these isolates. However, despitethe inability of some P. putida Fl::mer strains to utilizePMA, the remaining strains were able to do so. Thus,combining the activities of broad-spectrum Hgr with theendogenous degradative capabilities of P. putida Fl (11)generated a novel catabolic pathway in those isolates dem-onstrating PMA-utilization capabilities.

In summary, present efforts were aimed at constitutivelyoverexpressing mercury detoxification determinants to gen-erate highly (organo)mercurial-resistant bacterial strains ca-pable of detoxifying target compounds at increased rates andat elevated Hg concentrations. Overexpression wasachieved by random mutagenesis using a mini-TnS encodingmerTPAB. merA expression among ::mer isolates variedsignificantly (Table 2) and correlated with variations inresistance to PMA and Hg(II) (Fig. 2), and hybridizationanalysis indicated that only a single copy of merTPAB waspresent in each of the isolates tested (Fig. 1). These varia-tions between P. putida: :mer strains are thus consistent withupstream host promoters providing at least some merTPABtranscriptional activity, presumably by readthrough of the19-bp IS50 elements.The P. putida::mer isolates described here may have

potential for treatment of Hg-containing pollutants in bothcontained and environmental applications. Growth in higherconcentrations of mercuric compounds and especially con-stitutive expression of this capability enables survival inmore contaminated wastes before expression can be other-wise fully induced. Moreover, mercury detoxification wouldalso function when Hg(II) concentrations are subtoxic andtoo low to induce Hg(II) reduction in naturally occurringpopulations. The combination of organomercury resistanceand benzene degradation additionally permits detoxificationof mixed wastes and avoidance of cell toxicity by mineral-izing the benzene product of organomercurial lyase activityon phenylmercury.

We thank D. Gibson for providing P. putida Fl.This work was supported by the Ministry for Research and

Technology (BMFT) and the Fonds der Chemishe Industrie.

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